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Larval morphology of the scorpionfly Dicerapanorpa magna (Chou) (Mecoptera: Panorpidae) and its adaptive significance
Zoologischer Anzeiger 253 (2014) 216–224
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Larval morphology of the scorpionfly Dicerapanorpa magna (Chou) (Mecoptera: Panorpidae) and its adaptive significance Na Ma, Hongmin Chen 1 , Baozhen Hua ∗ Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China
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Article history: Received 3 September 2013 Received in revised form 22 October 2013 Accepted 28 October 2013 Available online 6 December 2013 Corresponding Editor: Sven Bradler. Keywords: Larvae Ultrastructure Mouthparts Chaetotaxy Adaptability
a b s t r a c t Larval morphology can provide valuable characters for taxonomic and phylogenetic analyses of insects and reflect the adaptations to various living habits. Compared with the adult stages, larval study has lagged far behind in Mecoptera. Although several genera of Panorpidae have been studied for their larval stages, the larva of Dicerapanorpa Zhong and Hua, 2013 basically remains unclear. Here the larva of Dicerapanorpa magna (Chou) is described and illustrated in detail for the first time using light microscopy and scanning electron microscopy. The larva is eruciform, with eight pairs of abdominal prolegs in addition to three pairs of thoracic legs, as in other Panorpidae. The most remarkable characteristics of the larvae include a pair of erect subdorsal annulated processes each on abdominal segments I–IX (A1–A9) and a single middorsal annulated process on A10, as well as a pair of prominent compound eyes composed of over 40 ommatidia, which distinguish this genus from other genera of Panorpidae. The annulated processes may have adaptive significance for fossorial and soil-living habits. © 2013 Elsevier GmbH. All rights reserved.
1. Introduction Larvae, a considerable part of the insect life cycle, are dramatically divergent in external morphology and dietary habits from adults in Endopterygota (Grimaldi and Engel, 2005; Van Emden, 1957; Yang, 2001; Zacharuk and Shields, 1991). Larval morphology reveals many informative diagnostic characters at various different taxonomic levels; some of them even provide more reliable phylogenetic information than adult morphology (Van Emden, 1957). Some studies contribute to resolve the phylogenetic relationships of some insect groups based on larval characters of especially medically or economically important insects in Diptera and Coleoptera (Alarie et al., 2011; Harbach and Kitching, 1998; Micó et al., 2008; Michat and Archangelsky, 2009; Nieves-Aldrey et al., 2005; Oosterbroek and Courtney, 1995; Pessoa et al., 2008; Reinert et al., 2008; Van Emden, 1957). Panorpidae, the largest family of Mecoptera, exhibit some peculiar features, such as the exposed upward genital bulb and the nuptial feeding mating behavior (Byers and Thornhill, 1983). The larvae of Panorpidae possess a pair of compound eyes on the lateral side of the head, unique among holometabolous insect groups
∗ Corresponding author. Tel.: +86 029 87091342; fax: +86 029 87091342. E-mail address: [email protected] (B. Hua). 1 Present address: Chisha Township Government in Chencang District, Baoji City, Shaanxi 721319, China. 0044-5231/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jcz.2013.10.002
(Byers, 1987; Chen et al., 2012; Paulus, 1979). Since panorpid larvae conceal themselves in soil and are difficult to observe, our knowledge on the group is largely confined to the adult stage (Cai and Hua, 2009a; Hua and Cai, 2009; Yie, 1951). Diceropanorpa Zhong and Hua, 2013 is endemic to China, with Panorpa magna Chou in Chou et al., 1981 as its type species and is recognizable mainly by two digitate anal horns on the tergum VI of males (Zhong and Hua, 2013). The larval stage of this genus has not been documented. We investigated the larval morphology and chaetotaxy of Dicerapanorpa magna (Chou in Chou et al., 1981) using light and scanning electron microscopy to provide additional evidence for phylogenetic analysis and explore the adaptive significance of some larval characters of Panorpidae.
2. Materials and methods Adults of D. magna were collected and reared in the Huoditang Forest Farm in the Qinling Mountains, Shaanxi Province of central China, from June to July 2010. The adults were reared in screen-wired cages (40 cm × 60 cm × 60 cm) and fed freshly killed flies, grasshoppers, katydids, or caterpillars. Live potted plants and wet absorbent cotton pads were provided in cages for simulating natural conditions. Gravid females were transferred to a covered transparent plastic cup containing 4 cm of humid soil for oviposition. Eggs were collected and incubated at room temperature (about 20–23 ◦ C) in plastic cups.
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Fig. 1. Light micrographs of the first instar larvae of D. magna. (A) Habitus. Arrow shows the prothoracic spiracle and arrowheads show the spiracles on abdomen. (B) Dorsal view of the head. (C) Lateral view of the head. (D) Ventral view of the head. Abbreviations: ac, anteclypeus; amc, anterior mandibular acetabulum; ant, antenna; apr, annulated process; at, anterior tentorial pits; cas, cardo-stipes; ce, compound eye; cly, clypeus; cs, coronal suture; eb, egg burster; epi, epipharynx; fcs, frontoclypeal sulcus; fs, frontal sutures; gl, galea; lbr, labrum; lp, labial palp; md, mandible; mp, maxillary palp; pm, postmentum; ppf; palpifer; prm, prementum; pt, posterior tentorial pit. Scale bars: (A) = 1 mm; (B)–(D) = 0.5 mm.
For light microscopy, larvae (n = 10) were killed by immersion in 60 ◦ C water to straighten and expand the body, and preserved in 75% ethanol. Photographs were taken with a QImaging Retiga 2000R Fast 1394 Digital CCD camera (QImaging, Surrey, BC, Canada) attached to a Nikon SMZ1500 Stereoscopic Zoom Microscope (Nikon, Tokyo, Japan). In order to examine the chaetotaxy in more detail, the larvae were decapitated and cut longitudinally along the midventral line, macerated in cold 4% NaOH for 24 h and rinsed with water several times. The integument was flattened and temporarily mounted in glycerin on a slide. Chaetotaxy was illustrated with the aid of a camera lucida attached to the microscope. The draft drawing was improved using Adobe Illustrator CS3, and the shapes of setae were modified based on details revealed in scanning electron micrographs. Chaetotaxy nomenclature primarily follows Hinton (1946) and Cai and Hua (2009a). For scanning electron microscopy (SEM), larvae of different instars (n = 8, 5, 6, 6, respectively, from the first to fourth instars) were fixed in Carnoy’s fixative solution (95% ethanol: glacial acetic acid = 3:1, v/v) for 12 h and preserved in 75% ethanol. The samples were dehydrated in a graded ethanol series and transferred to isoamyl acetate twice for 30 min, critical point-dried with liquid CO2 (HCP-2 Critical Point Dryer, Hitachi, Tokyo, Japan), sputtercoated with gold (JFC-1600 Auto Fine Coater, JEOL, Tokyo, Japan), and observed in a JEOL JSM-6360LV scanning electron microscope (JEOL, Tokyo, Japan).
The head of the larva is yellowish-brown and well-sclerotized, with an average width of 0.71 mm in the first instar and 1.70 mm in the fourth instar larvae (Figs. 1 and 2A). The coronal and frontal sutures join together to form an inverted Y-shaped ecdysial line, with the stem medially extending from the occipital foramen and the lateral arms diverging downward to the frontoclypeal suture (Figs. 1B and 2A). The egg burster, a sharp triangular projection that aids in hatching of the larva, is situated medially close to the frontoclypeal suture in the first instar (Fig. 1B) and disappears after the first molt. The anterior tentorial pits are located at the lateroventral margin of the intersection of the frontal suture and frontoclypeal suture (Figs. 1B and 2A). The posterior tentorial pits lie ventrally near the occipital sulcus (Figs. 1D and 2C). Thirteen pairs of setae are distributed on the cranium symmetrically. The region between the eye and gena is covered with numerous minute granules in the first instar, but nearly glabrous in other three instars (Fig. 2A and B). The trapezoid clypeus is separated from the frons by the distinct frontoclypeal suture and subdivided into the basal sclerotized postclypeus and the apical membranous anteclypeus by a slightly sunken line with a transverse row of four setae (Figs. 1B, 2A and D).
3. Results
3.3. The compound eyes and antennae
3.1. General morphology of the larva
The compound eyes are hemispherical and purplish-red, situated dorsolaterally between the vertex and genae. Each eye is composed of over 40 ommatidia, with the peripheral ommatidia paler in color and blurry in margin (Figs. 1C and 2B). The paired antennae each are located between the compound eye and clypeus (Fig. 2B), and consist of three segments: a basal scape, a pedicel, and a distal flagellum (Fig. 3). The scape is inserted into a membranous antennal socket supported by a raised antennal sclerite (Fig. 3B and C). The pedicel is the longest segment and expanded, with a number of cup-like depressions with small granular central processes arranged in two opposed groups on the inner
The larva is eruciform, with eight pairs of abdominal prolegs in addition to three pairs of thoracic legs (Fig. 1A). The antennae are 3-segmented (Fig. 1B). The mouthparts are of the mandibulate type. A pair of prominent compound eyes is situated dorsolaterally between the vertex and gena (Fig. 1C). The abdominal segments are equipped with paired erect subdorsal annulated processes on terga I–IX and a single middorsal annulated process on tergum X. The respiratory system is peripneustic, with one prothoracic, and eight abdominal spiracles on the pleura of the prothorax and first eight
abdominal segments, respectively (Fig. 1A). The larvae undergo four instars, which are distinct in size but similar in gross morphology. 3.2. The head capsule
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Fig. 2. SEM micrographs of the larval heads of D. magna. (A) Dorsal view, fourth instar. (B) Lateral view, first instar. (C) Ventral view, fourth instar. (D) Magnification of the mouthparts of fourth instar. Abbreviations: ac, anteclypeus; amc, anterior mandibular acetabulum; ant, antenna; at, anterior tentorial pits; cas, cardo-stipes; ce, compound eye; cly, clypeus; cs, coronal suture; eb, egg burster; epi, epipharynx; fcs, frontoclypeal sulcus; fs, frontal sutures; gl, galea; lbr, labrum; lp, labial palp; md, mandible; mp, maxillary palp; pc, postclypeus; pm, postmentum; ppf; palpifer; prm, prementum; pt, posterior tentorial pit. Scale bars: (A) and (C) = 500 m; (B) and (D) = 200 m.
Fig. 3. Antenna of D. magna larvae, SEM micrographs. (A) First instar, latero-ventral view. (B) Second instar, ventral view. (C) Fourth instar, dorsal view. (D) Magnification of the pedicel of fourth instar larva in ventral view, showing the sensory pits on the apical half. Abbreviations: as, antennal sclerite; bs, basiconic sensilla; fl, flagellum; pe, pedicel; sc, scape; sp, sensory pits. Scale bars: (A) and (D) = 20 m; (B) = 50 m; (C) = 100 m.
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Fig. 4. Eipharynx of D. magna larvae, SEM micrographs. (A) First instar larva. (B) Third instar larva. (C) Fourth instar larva. (D) Magnification of the apical part of the epipharynx of first instar larva. Abbreviations: bs, basiconic sensilla; lr, labral setae; ps, palmate sensilla. Scale bars: (A) = 50 m; (B) and (C) = 100 m; (D) = 20 m.
and ventral sides of the apical third (Fig. 3A, B and D). The number of depressions varies in different instars, approximately 20 in the first instar and 30 in the fourth instar (Fig. 3A and D). The flagellum is considerably thin, shorter than the pedicel, with several sensilla basiconica at apex. The dorsal surface of the antenna is glabrous, lacking sensilla (Fig. 3C). 3.4. The mouthparts The mouthparts are of the typical mandibulate type. The labrum is trapezoid and slightly notched midapically, and articulated proximally with the anterior region of the clypeus (Fig. 2A and D). The labrum is furnished with two pairs of long labral setae along the distal margin, the inner pair nearly two-thirds the length of the outer pair (Fig. 2A). The membranous epipharynx is situated on the inner surface of the labrum and is basically in accordance with the labrum in shape (Fig. 4). At the mid-apical region appears a distinct longitudinal furrow (Fig. 4A–C). The epipharynx is furnished with dense lateral microtrichia pointed inward and sparse short medial spines pointed upward, with the apicolateral corner and longitudinal furrow regions glabrous (Fig. 4A–C). On the epipharynx, two types of sensilla are arranged symmetrically: five pairs of basiconic sensilla at the apical margin (three pairs) and between the microtrichial and spinous regions (two pairs), and two pairs of palmate sensilla near the basolateral microtrichial region (Fig. 4). A triangular sclerite connecting the epipharynx with the clypeus is enclosed with rows of spines. Several basiconic sensilla are observed at the basal and apical parts (Fig. 4A–C). The middle region enclosed by the spines is rugose and nearly glabrous. A few short microtrichia are extended along the midline of the triangular sclerite. The middle microtrichia nearly extend to two-thirds of the midline in the first instar, but do not reach the first third in the third and fourth instars (Fig. 4A–C). The paired mandibles are strongly sclerotized and curve inward, crossing each other apically and uniting to the subgena at the clypeal base (Fig. 2D). They are typically dicondylic, with a notched anterior ginglymus and knob-like posterior condyle (Fig. 5A–D).
The mandible tapers toward the apex, with two long subequal setae and one short seta on the lateral surface (Fig. 5B). The incisor region bears an acuminate triangular tooth, as well as a median and two basal teeth (Fig. 5A–D). The median tooth is longer than the basal teeth, but much shorter than the apical tooth. The molar region is equipped with numerous regularly arranged conical teeth, which are bordered by a row of spines on each lateral side (Fig. 5A–E). A tuft of branched microtrichia subtends the base of the molar region (Fig. 5E). In the newly hatched larva, the molar teeth and spines are sharp and intact, but become worn down after feeding (Fig. 5F). The paired maxillae each consist of the basal cardo-stipes, galea, lacinia, and three-segmented maxillary palp (Figs. 1D, 2C and 6A). The cardo-stipes is kidney-shaped and distally connected to the galea and lacinia medially and bears the palp laterally. Two setae are located on the cardo-stipes and palpifer, respectively (Figs. 1D and 2C). The galea is sclerotized in the basal half and membranous in the apical half, which is covered with dense microtrichia in the posterior side. The anterior surface of galea is equipped with numerous branched microtrichia, among which are distributed several basiconic sensilla at the apical half. The galea is broadly connected with the lacinia at base, the glabrous joint region bearing one palmate sensillum and two campaniform sensilla (Fig. 6A and B). The lacinia is considerably small, furnished with regularly arranged microplates at the apical half and dense microtrichia at the basal half. The three-segmented maxillary palp is inserted on a sclerotized palpifer, with the basal two segments roughly equal in length and shorter than the distal segment (Fig. 6A and B). The distal segment bears 14 basiconic sensilla on the apex (Fig. 6C). The labium is greatly reduced in size, with ligula absent. Most parts of the labium are retracted into the capsule; only a pair of two-segmented labial palps are prominently visible from the basal, mesially separated prementum between the cardo-stipes bases of the paired maxillae. Located at the base between the divided prementum is the opening of the salivary duct (Fig. 6D). The distal segment of the labial palp is much longer than the basal segment, approximately three times the length of the latter, and bears ten basiconic sensilla on its apex (Fig. 6D).
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Fig. 5. Mandibles of D. magna larvae, SEM micrographs. (A) Posterior view of the right mandible of first instar. (B) Anterior view of the left mandible of first instar. (C) Fronto-lateral view of the right mandible of first instar. (D) Fronto-lateral view of the left mandible of third instar. (E) Regular spines along the molar region and branched microtrichia at the base, first instar. (F) Molar teeth and spines worn down in fourth instar. Abbreviations: ant, antenna; c, condyle; g, ginglymus; ir, incisor region; mr, molar region. Scale bars: (A)–(C) and (F) = 50 m; (D) = 100 m; (E) = 20 m.
3.5. The thorax The prothorax is different from the meso- and metathorax by its prothoracic shield, paired spiracles, and chaetotaxy (Fig. 7A). The prothoracic shield is broad and saddle-like, with several short setae along the margin. The spiracle is situated at the posterolateral corner of the prothoracic shield and consists of a central atrial orifice and 12 peripheral apertures (Fig. 7A and C). Meso- and metathorax are similar in morphology and chaetotaxy, and lacking spiracles. The thoracic legs are 4-segmented: the coxa, femur, tibia and tarsus (Fig. 7D). The basal coxa and femur furnished with numerous microtrichia and long setae represent the thickest and longest segments, respectively. The apical halves of both the tibia and tarsus are hirsute (Fig. 7D). 3.6. The abdomen The abdomen consists of 11 segments and is furnished with short setae on the pinacula and erect subdorsal annulated processes inserted on stout basal protuberances (Fig. 1A). The long annulated processes are paired on segments I–IX (A1–A9), but single on the middorsal line of A10 (Figs. 1A and 7B). On the annulated process, numerous scaly cuticular processes are palmated. A campaniform sensillum is located at the base of the annulated process
(Fig. 7B). The first seven abdominal segments are roughly similar to each other in morphology and chaetotaxy (Fig. 1A). A8–A10 have much longer processes, and are thinner and differ considerably from others in chaetotaxy (Fig. 7B). Paired lateral spiracles and ventral prolegs are located on the first eight abdominal segments (Figs. 1A and 7B). The spiracle consists of seven spherical apertures (Fig. 7E), less than that of the prothoracic spiracle. The stout prolegs are unsegmented and are not arranged in a longitudinal line with the thoracic legs (Fig. 7F). A11 is reduced and remains as a four-lobed protrusile sucker (Fig. 7B). 3.7. The chaetotaxy of trunk The chaetotaxy of the mesothorax is similar to that of the metathorax, but different from that of the prothorax. For the abdomen, the first seven segments bear similar chaetotaxy, while segments VIII–X are individually distinct. The chaetotaxy of the trunk is illustrated in Fig. 8. Prothorax (T1): Along the anterior edge of the prothoracic shield are three clavate setae (XD1, XD2 and SD2, about 86, 104 and 99 m in length, respectively) and three punctures. Near the middorsal line appear a short setiform seta (D2) and a puncture. Two setae (D1 and SD1) are along the posterior edge of the shield. D1 is above the level of D2 and longer than half length of SD1. The distance
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Fig. 6. Maxilla and labium of D. magna larvae, SEM micrographs. (A) Maxilla of first instar, posterior view. Inset shows the magnification of the palmate sensillum. (B) Maxilla of third instar, showing the medial surface of the maxillary palp. Inset shows the magnification of one campaniform sensillum. (C) Magnification of the distal maxillary palp in first instar. (D) Labium region of first instar, showing the two-segmented labial palp. Abbreviations: bs, basiconic sensilla; cas, cardo-stipes; cs, campaniform sensilla; gl, galea; lc, lacinia; lp, labial palp; mp, maxillary palp; pm, postmentum; ppf; palpifer; prm, prementum; ps, palmate sensilla; sdo, salivary duct opening. Scale bars: (A) and (B) = 50 m; (C) = 10 m; (D) = 20 m.
between XD2 and SD2 is slightly shorter than that between D1 and SD1. L1 is on the lateral pinaculum, anteroventral to the spiracle. Two long setae (SV1 and SV2, 107 and 58 m long, respectively) and two microsetae (MSV1 and MSV2) on a crescent pinaculum dorsal to the coxal cavity. A spiculate ventral seta (V1) is close to the midventral line. Meso- and metathorax (T2 and T3): On the trapezoid dorsal pinaculum are three clavate setae (D1, D2, and SD1) and two microsetae (MD1 and MSD1). Ventral to the dorsal pinaculum are three pinacula, each of which is accompanied respectively by a long seta (SD2, L1, and L2) and a microseta (MSD2, ML1, and ML2). Subventral setae (SV1 and SV2) are located on two detached subventral pinacula. Ventral to the coxal cavity are one spiculate seta (V1) and a pair of microsetae (MV1 and MV2). Among these setae, SD1 is roughly 123 m long; longer than twice the length of D1. SD2 is slightly longer than L1, which is three-quarters as long as L2 (122 m). SV2 is nearly three-fourths the length of SV1. Abdominal segments I–VII (A1–A7): The annulated processes on A1–A7 measure 306 m in length on average. On the dorsal pinaculum are three rhabdiform setae (D1, D2, and SD1) and two microsetae (MD1 and MSD1). SD2 is accompanied by two microsetae (MSD2 and MSD3). Posterior to the spiracle is a long lateral seta (L1) with two microsetae (ML1 and ML3). L2 is located together with ML2 on a lower lateral pinaculum. Three setae (SV1–SV3) are on two subventral pinacula. Paired ventral setae (V1 and V2) are setiform, anterior to the proleg. D2 is almost equal to D1 in length. SD1 and SD2 are roughly in the same length (112 m on average), slightly shorter than L1. SV2 and SV3 are half as long as SV1. Abdominal segment VIII (A8): Three setae on the stout basal protuberance of the annulated process are rhabdiform, 41–42 m long for the lateral and mesal setae and 63 m for the posterior seta. L1 and L2 are rhabdiform. SV1–SV3 taper apically. L1 is about 128 m long, 1.6 times as long as L2 and markedly shorter than SV1. The setae between the spiracle and midventral line are identical to the preceding abdominal segments.
Abdominal segment IX (A9): Three setae on the basal protuberance of the annulated process are arranged similar to those on A8. D2, SD1, and SD2 are almost in the same length. L1 and SV1 are setiform, almost equal in length (173 m). V1 is about 63 m long. Abdominal segment X (A10): The protuberance of the single middorsal annulated process bears only two setae, each on lateral and mesal sides. A palmate pinaculum is located from the dorsal side to the subventral side, with one long, one short, and one minute seta at its posterior corner, regressively and four digitate lateral processes each terminated by a microseta. Setae D2, SD1 and SD2 are acicular, similar in length. 4. Discussion Larval data play an important role in the reconstruction of phylogenies in holometabolous insects, including Mecoptera (Beutel et al., 2009; Fraulob et al., 2012; Friedrich et al., 2013). In Panorpidae, although some species of Panorpa, Neopanorpa and Sinopanorpa have been described for their larval stages (Brauer, 1863; Byers, 1954, 1963, 1987; Cai and Hua, 2009b; Chen and Hua, 2011; Gassner, 1963; Mampe and Neunzig, 1965; Miyaké, 1912; Potter, 1938; Steiner, 1930; Yie, 1951), this paper is the first attempt to describe and illustrate the larva of the genus Dicerapanorpa. Panorpid larvae are peculiar among the holometabolous larvae for the presence of paired compound eyes (Byers, 1987). According to previous investigations, a compound eye comprises 24–35 ommatidia in Neopanorpa and Panorpa (Boese, 1973; Byers, 1963, 1987; Cai and Hua, 2009a,b; Steiner, 1930; Yie, 1951) and does not increase in the number of ommatidia during development, although differing among genera or species (Chen and Hua, 2011; Paulus, 1989; Yie, 1951). Based on our observations, the compound eye of D. magna larva is composed of over 40 ommatidia, similar to that of Sinopanorpa (Chen and Hua, 2011). The mandibulate mouthparts of panorpid larvae show slight differences among genera. For example, the labral apex is set with
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Fig. 7. Trunk of the first instar larvae of D. magna, SEM micrographs. (A) Lateral view of the thorax. (B) Lateral view of the abdominal segments VI to telson. Inset shows the magnification of the annulated process and setae on the abdominal segment IX. (C) Prothoracic spiracle. (D) Thoracic legs, lateral view. (E) Abdominal spiracle. (F) Prolegs of abdominal segments III and IV (A3 and A4), lateral view. Abbreviations: A, abdominal segment; ao, atrial orifice; ap, aperture; apr, annulated process; cs, campaniform sensillum; cx, coxa; fm, femur; pl, proleg; sp, spiracle; T, thorax; tb, tibia; tl, telson; ts, tarsus. Scale bars: (A), (B), (D) and (F) = 100 m; (C) and (E) = 10 m.
paired long setae laterally and one pair of short setae medially in Neopanorpa. In addition to the lateral paired long setae, two pairs of short setae are observed in D. magna and Sinopanorpa tincta and two or three pairs in Panorpa (Chen and Hua, 2011; Yie, 1951). According to Yie (1951), both the maxillary and labial palps bear two sensory cavities and several minute setae on the apices of the distal segments. Under SEM, we found the minute setae are in fact basiconic sensilla but the two sensory cavities were not observed. As gustatory chemoreceptors, the basiconic sensilla have a great ability to determine acceptable food items (Zacharuk and Shields, 1991). The number of basiconic sensilla on the apices of both maxillary and labial palps varies among different genera and species of Panorpidae. On the apex of the distal segment of the maxillary palp, 14 basiconic sensilla are present in D. magna, three more than that of Sinopanorpa, four more than that of Neopanorpa, and 6–8 more than that of Panorpa (Cai and Hua, 2009a,b; Chen and Hua, 2011; Yie, 1951). With respect to the segment number of labial palp in Panorpidae, controversial opinions are present. Bierbrodt (1942) regarded the labial palp as three-segmented based on the palpal muscles in Panorpa, while Hinton (1958) considered that the muscles referred by Bierbrodt (1942) were actually inserted on the lobed prementum and the labial palp was two-segmented. According to Snodgrass (1935), the prementum represents the united stipes of a pair of
maxilla-like appendages and is suggested often by a distal cleft between its stipital components. Our SEM observation supports Hinton (1958) with respect to the distally cleft prementum and the two-segmented labial palps. The distal segment of the labial palp bears apically ten basiconic sensilla in D. magna and S. tincta, eight or ten in Neopanorpa, and only six in Panorpa. The divided clypeal region of mecopteran larvae has been described in genera Panorpa and Sinopanorpa of Panorpidae (Bierbrodt, 1942; Chen and Hua, 2011), Apterobittacus, Harpobittacus, and Bittacus of Bittacidae (Applegarth, 1939; Currie, 1932; Tan and Hua, 2008), and in Boreus of Boreidae and Nannochorista of Nanonchoristidae (Beutel et al., 2009). Compared with the narrow and elongated anteclypeus in Bittacidae and other genera of Panorpidae, the anteclypeus in Dicerapanorpa is broader, with a distinct transverse demarcation, similar to that of Nannochorista (Beutel et al., 2009). Dicerapanorpa larvae can be readily distinguished from other genera of Panorpidae by the following characters: (i) the compound eye is composed of more than 40 ommatidia; (ii) the numbers of the basiconic sensilla on the apices of both maxillary and labial palps are 14 and 10, respectively; (iii) the setae are short and annulated processes are longer; (iv) MSD1 on the prothorax is located almost along the same horizontal line with SD2; and (v) microtrichia are lacking posterior to SD2 on A10.
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Fig. 8. Chaetotaxy of the trunk of D. magna, first instar. Abbreviations: A, abdomen; D, dorsal setae; L, lateral setae; MD, minute dorsal seta; ML, minute lateral setae; MSD, minute subdorsal setae; MSV, minute subventral seta; MV, minute ventral setae; pl, proleg; SD, subdorsal setae; sp, spiracle; SV, subventral setae; T, thorax; V, ventral setae; XD, prothoracic setae. Scale bar = 0.1 mm.
The abdomen of D. magna larvae terminates in a four-lobed protrusile sucker, which is similar to that of the rectal processes of other eruciform larvae of Pistillifera, but may not be homologous with the anal papillae in Nannochorista because of the very farreaching structural and functional differences (Beutel et al., 2009; Pilgrim, 1972). According to embryonic evidence, the protrusile sucker of Panorpa is developed from the processes of the eleventh abdominal segment (Du et al., 2009). Hence, the reduced A11 in Panorpidae may be evolved secondarily, similar to the remarkable A11 in Boreidae (Byers, 1987; Russell, 1982). Mecoptera exhibit remarkable morphological diversity in their larval stage, and adapted to various habitats. The larvae of the primitive Nannochoristidae are campodeiform, with their body surface almost smooth and shining, adapted to aquatic habits (Fraulob et al., 2012; Pilgrim, 1972). The larvae of Boreidae and Panorpodidae were regarded as scarabaeiform, and only bear short setae on their trunks, relating to tunneling into the sod of host mosses and substrate, respectively (Byers, 1987, 1997; Cooper, 1974; Penny, 1977; Peterson, 1951; Potter, 1938; Russell, 1982; Suzuki, 1990). The larvae of Bittacidae and Panorpidae are eruciform and bear prominent protuberances on their trunk (Byers, 1987). The protuberances
in Bittacidae have two or three branches from which setae arise (Applegarth, 1939; Currie, 1932; Setty, 1939, 1940; Suzuki, 1990; Tan and Hua, 2008, 2009), but the protuberances in Panorpidae each bear a long annulated process (Brauer, 1863; Byers, 1954, 1963; Cai and Hua, 2009b; Chen and Hua, 2011; Gassner, 1963; Mampe and Neunzig, 1965; Miyaké, 1912; Potter, 1938; Steiner, 1930; Yie, 1951). The newly hatched larvae of Bittacidae do not burrow into soil and usually cover their body surfaces with mud, probably for camouflage (Tan and Hua, 2008). However, the larvae of Panorpidae burrow through the soil shallowly (approximately 2.5–7.5 cm) to forage beneath potential food. When the food is situated over 7.5 cm away, they crawl above the substrate until food is located, and then constructs a new burrow for concealment while feeding (Mampe and Neunzig, 1965). We speculate that the occurrence of the long annulated processes on the larval trunk of Panorpidae is associated with the above-substrate searching and fossorial feeding habits, and is very likely evolved as adaptations to protect the soft body from much leaf litter. The dense palmate cuticular processes of the annulated process may reduce friction when the larvae move forward either above or below the substrate.
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Acknowledgements We thank Tao Li for assistance in larval rearing. We also thank Louis A. Somma and an anonymous reviewer for critically reviewing our manuscript. This research was supported by the National Natural Science Foundation of China (Grant No. 31172125) for B.Z. Hua and the China Postdoctoral Science Foundation (Grant Nos. 2012M520085 and 2013T60893) for N. Ma. References Alarie, Y., Short, A.E.Z., Garcia, M., Joly, L., 2011. Larval morphology of Meruidae (Coleoptera: Adephaga) and its phylogenetic implications. Annals of the Entomological Society of America 104, 25–36. Applegarth, A.G., 1939. The larva of Apterobittacus apterus MacLachlan (Mecoptera: Panorpidae). Microentomology 4, 109–120. Beutel, R.G., Kristensen, N.P., Pohl, H., 2009. Resolving insect phylogeny: the significance of cephalic structures of the Nannomecoptera in understanding endopterygote relationships. Arthropod Structure & Development 38, 427–460. Bierbrodt, E., 1942. Der Larvenkopf von Panorpa communis L. und seine Verwandlung, mit besonderer Berücksichtigung des Gehirns und der Augen. Zoologische Jahrbücher Abteilung für Anatomie und Ontogenie der Tiere 68, 49–136. Boese, A.E., 1973. Descriptions of larvae and key to fourth instars of North American Panorpa (Mecoptera: Panorpidae). University of Kansas Science Bulletin 50, 163–186. Brauer, F., 1863. Beitrage zur Kenntniss der Panorpiden-Larven. Verhandlungen der Kaiserlich-Koniglichen Zoologisch-Botanischen Gesellschaft in Wien 13, 307–324. Byers, G.W., 1954. Notes on North American Mecoptera. Annals of the Entomological Society of America 47, 484–510. Byers, G.W., 1963. The life history of Panorpa nuptialis (Mecoptera: Panorpidae). Annals of the Entomological Society of America 56, 142–149. Byers, G.W., 1987. Order Mecoptera. In: Stehr, F.W. (Ed.), Immature Insects. Kendall/Hunt Publishing, Dubuque, pp. 246–252. Byers, G.W., 1997. Biology of Brachypanorpa (Mecoptera: Panorpodidae). Journal of the Kansas Entomological Society 70, 313–322. Byers, G.W., Thornhill, R., 1983. Biology of the Mecoptera. Annual Review of Entomology 28, 203–228. Cai, L.J., Hua, B.Z., 2009a. Morphology of the immature stages of Panorpa qinlingensis (Mecoptera: Panorpidae) with notes on its biology. Entomologica Fennica 20, 215–224. Cai, L.J., Hua, B.Z., 2009b. A new Neopanorpa (Mecoptera: Panorpidae) from China with notes on its biology. Deutsche Entomologische Zeitschrift 56, 93–99. Chen, H.M., Hua, B.Z., 2011. Morphology and chaetotaxy of the first instar larva of the scorpionfly Sinopanorpa tincta (Mecoptera: Panorpidae). Zootaxa 2897, 18–26. Chen, Q.X., Li, T., Hua, B.Z., 2012. Ultrastructure of the larval eye of the scorpionfly Panorpa dubia (Mecoptera: Panorpidae) with implications for the evolutionary origin of holometabolous larvae. Journal of Morphology 273, 561–571. Chou, I., Ran, R.B., Wang, S.M., 1981. Taxonomic study on the Chinese Mecoptera (I, II). Entomotaxonomia 3, 1–22. Cooper, K.W., 1974. Sexual biology, chromosomes, development, life histories and parasites of Boreus, especially of B. notoperates. A southern California Boreus. II (Mecoptera: Boreidae). Psyche 81, 84–120. Currie, G.A., 1932. Some notes on the biology and morphology of the immature stages of Harpobittacus tillyardi (Order Mecoptera). Proceedings of the Linnean Society of New South Wales 57, 116–122. Du, X.L., Yue, C., Hua, B.Z., 2009. Embryonic development of the scorpionfly Panorpa emarginata Cheng with special reference to external morphology (Mecoptera: Panorpidae). Journal of Morphology 270, 984–995. Fraulob, M., Wipfler, B., Hunefeld, F., Pohl, H., Beutel, R.G., 2012. The larval abdomen of the enigmatic Nannochoristidae (Mecoptera, Insecta). Arthropod Structure & Development 41, 187–198. Friedrich, F., Pohl, H., Beckmann, F., Beutel, R.G., 2013. The head of Merope tuber (Meropeidae) and the phylogeny of Mecoptera (Hexapoda). Arthropod Structure & Development 42, 69–88. Gassner, G., 1963. Notes on the biology and immature stages of Panorpa nuptialis (Mecoptera: Panorpidae). Texas Journal of Science 15, 142–154. Grimaldi, D., Engel, M.S., 2005. Evolution of the Insects. Cambridge University Press, Cambridge. Harbach, R.E., Kitching, I.J., 1998. Phylogeny and classification of the Culicidae (Diptera). Systematic Entomology 23, 327–370. Hinton, H.E., 1946. On the homology and nomenclature of the setae of lepidopterous larvae, with some notes on the phylogeny of the Lepidoptera. Transactions of the Royal Entomological Society of London 97, 1–37.
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Dicerapanorpa, a new genus of East Asian Panorpidae (Insecta: Mecoptera: Penorpidae) with descriptions of two new species. Journal of Natural History 47, 1019–1046.
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Larval morphology of the scorpionfly Dicerapanorpa magna (Chou) (Mecoptera: Panorpidae) and its adaptive significance Na Ma, Hongmin Chen 1 , Baozhen Hua ∗ Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China
a r t i c l e
i n f o
Article history: Received 3 September 2013 Received in revised form 22 October 2013 Accepted 28 October 2013 Available online 6 December 2013 Corresponding Editor: Sven Bradler. Keywords: Larvae Ultrastructure Mouthparts Chaetotaxy Adaptability
a b s t r a c t Larval morphology can provide valuable characters for taxonomic and phylogenetic analyses of insects and reflect the adaptations to various living habits. Compared with the adult stages, larval study has lagged far behind in Mecoptera. Although several genera of Panorpidae have been studied for their larval stages, the larva of Dicerapanorpa Zhong and Hua, 2013 basically remains unclear. Here the larva of Dicerapanorpa magna (Chou) is described and illustrated in detail for the first time using light microscopy and scanning electron microscopy. The larva is eruciform, with eight pairs of abdominal prolegs in addition to three pairs of thoracic legs, as in other Panorpidae. The most remarkable characteristics of the larvae include a pair of erect subdorsal annulated processes each on abdominal segments I–IX (A1–A9) and a single middorsal annulated process on A10, as well as a pair of prominent compound eyes composed of over 40 ommatidia, which distinguish this genus from other genera of Panorpidae. The annulated processes may have adaptive significance for fossorial and soil-living habits. © 2013 Elsevier GmbH. All rights reserved.
1. Introduction Larvae, a considerable part of the insect life cycle, are dramatically divergent in external morphology and dietary habits from adults in Endopterygota (Grimaldi and Engel, 2005; Van Emden, 1957; Yang, 2001; Zacharuk and Shields, 1991). Larval morphology reveals many informative diagnostic characters at various different taxonomic levels; some of them even provide more reliable phylogenetic information than adult morphology (Van Emden, 1957). Some studies contribute to resolve the phylogenetic relationships of some insect groups based on larval characters of especially medically or economically important insects in Diptera and Coleoptera (Alarie et al., 2011; Harbach and Kitching, 1998; Micó et al., 2008; Michat and Archangelsky, 2009; Nieves-Aldrey et al., 2005; Oosterbroek and Courtney, 1995; Pessoa et al., 2008; Reinert et al., 2008; Van Emden, 1957). Panorpidae, the largest family of Mecoptera, exhibit some peculiar features, such as the exposed upward genital bulb and the nuptial feeding mating behavior (Byers and Thornhill, 1983). The larvae of Panorpidae possess a pair of compound eyes on the lateral side of the head, unique among holometabolous insect groups
∗ Corresponding author. Tel.: +86 029 87091342; fax: +86 029 87091342. E-mail address: [email protected] (B. Hua). 1 Present address: Chisha Township Government in Chencang District, Baoji City, Shaanxi 721319, China. 0044-5231/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jcz.2013.10.002
(Byers, 1987; Chen et al., 2012; Paulus, 1979). Since panorpid larvae conceal themselves in soil and are difficult to observe, our knowledge on the group is largely confined to the adult stage (Cai and Hua, 2009a; Hua and Cai, 2009; Yie, 1951). Diceropanorpa Zhong and Hua, 2013 is endemic to China, with Panorpa magna Chou in Chou et al., 1981 as its type species and is recognizable mainly by two digitate anal horns on the tergum VI of males (Zhong and Hua, 2013). The larval stage of this genus has not been documented. We investigated the larval morphology and chaetotaxy of Dicerapanorpa magna (Chou in Chou et al., 1981) using light and scanning electron microscopy to provide additional evidence for phylogenetic analysis and explore the adaptive significance of some larval characters of Panorpidae.
2. Materials and methods Adults of D. magna were collected and reared in the Huoditang Forest Farm in the Qinling Mountains, Shaanxi Province of central China, from June to July 2010. The adults were reared in screen-wired cages (40 cm × 60 cm × 60 cm) and fed freshly killed flies, grasshoppers, katydids, or caterpillars. Live potted plants and wet absorbent cotton pads were provided in cages for simulating natural conditions. Gravid females were transferred to a covered transparent plastic cup containing 4 cm of humid soil for oviposition. Eggs were collected and incubated at room temperature (about 20–23 ◦ C) in plastic cups.
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Fig. 1. Light micrographs of the first instar larvae of D. magna. (A) Habitus. Arrow shows the prothoracic spiracle and arrowheads show the spiracles on abdomen. (B) Dorsal view of the head. (C) Lateral view of the head. (D) Ventral view of the head. Abbreviations: ac, anteclypeus; amc, anterior mandibular acetabulum; ant, antenna; apr, annulated process; at, anterior tentorial pits; cas, cardo-stipes; ce, compound eye; cly, clypeus; cs, coronal suture; eb, egg burster; epi, epipharynx; fcs, frontoclypeal sulcus; fs, frontal sutures; gl, galea; lbr, labrum; lp, labial palp; md, mandible; mp, maxillary palp; pm, postmentum; ppf; palpifer; prm, prementum; pt, posterior tentorial pit. Scale bars: (A) = 1 mm; (B)–(D) = 0.5 mm.
For light microscopy, larvae (n = 10) were killed by immersion in 60 ◦ C water to straighten and expand the body, and preserved in 75% ethanol. Photographs were taken with a QImaging Retiga 2000R Fast 1394 Digital CCD camera (QImaging, Surrey, BC, Canada) attached to a Nikon SMZ1500 Stereoscopic Zoom Microscope (Nikon, Tokyo, Japan). In order to examine the chaetotaxy in more detail, the larvae were decapitated and cut longitudinally along the midventral line, macerated in cold 4% NaOH for 24 h and rinsed with water several times. The integument was flattened and temporarily mounted in glycerin on a slide. Chaetotaxy was illustrated with the aid of a camera lucida attached to the microscope. The draft drawing was improved using Adobe Illustrator CS3, and the shapes of setae were modified based on details revealed in scanning electron micrographs. Chaetotaxy nomenclature primarily follows Hinton (1946) and Cai and Hua (2009a). For scanning electron microscopy (SEM), larvae of different instars (n = 8, 5, 6, 6, respectively, from the first to fourth instars) were fixed in Carnoy’s fixative solution (95% ethanol: glacial acetic acid = 3:1, v/v) for 12 h and preserved in 75% ethanol. The samples were dehydrated in a graded ethanol series and transferred to isoamyl acetate twice for 30 min, critical point-dried with liquid CO2 (HCP-2 Critical Point Dryer, Hitachi, Tokyo, Japan), sputtercoated with gold (JFC-1600 Auto Fine Coater, JEOL, Tokyo, Japan), and observed in a JEOL JSM-6360LV scanning electron microscope (JEOL, Tokyo, Japan).
The head of the larva is yellowish-brown and well-sclerotized, with an average width of 0.71 mm in the first instar and 1.70 mm in the fourth instar larvae (Figs. 1 and 2A). The coronal and frontal sutures join together to form an inverted Y-shaped ecdysial line, with the stem medially extending from the occipital foramen and the lateral arms diverging downward to the frontoclypeal suture (Figs. 1B and 2A). The egg burster, a sharp triangular projection that aids in hatching of the larva, is situated medially close to the frontoclypeal suture in the first instar (Fig. 1B) and disappears after the first molt. The anterior tentorial pits are located at the lateroventral margin of the intersection of the frontal suture and frontoclypeal suture (Figs. 1B and 2A). The posterior tentorial pits lie ventrally near the occipital sulcus (Figs. 1D and 2C). Thirteen pairs of setae are distributed on the cranium symmetrically. The region between the eye and gena is covered with numerous minute granules in the first instar, but nearly glabrous in other three instars (Fig. 2A and B). The trapezoid clypeus is separated from the frons by the distinct frontoclypeal suture and subdivided into the basal sclerotized postclypeus and the apical membranous anteclypeus by a slightly sunken line with a transverse row of four setae (Figs. 1B, 2A and D).
3. Results
3.3. The compound eyes and antennae
3.1. General morphology of the larva
The compound eyes are hemispherical and purplish-red, situated dorsolaterally between the vertex and genae. Each eye is composed of over 40 ommatidia, with the peripheral ommatidia paler in color and blurry in margin (Figs. 1C and 2B). The paired antennae each are located between the compound eye and clypeus (Fig. 2B), and consist of three segments: a basal scape, a pedicel, and a distal flagellum (Fig. 3). The scape is inserted into a membranous antennal socket supported by a raised antennal sclerite (Fig. 3B and C). The pedicel is the longest segment and expanded, with a number of cup-like depressions with small granular central processes arranged in two opposed groups on the inner
The larva is eruciform, with eight pairs of abdominal prolegs in addition to three pairs of thoracic legs (Fig. 1A). The antennae are 3-segmented (Fig. 1B). The mouthparts are of the mandibulate type. A pair of prominent compound eyes is situated dorsolaterally between the vertex and gena (Fig. 1C). The abdominal segments are equipped with paired erect subdorsal annulated processes on terga I–IX and a single middorsal annulated process on tergum X. The respiratory system is peripneustic, with one prothoracic, and eight abdominal spiracles on the pleura of the prothorax and first eight
abdominal segments, respectively (Fig. 1A). The larvae undergo four instars, which are distinct in size but similar in gross morphology. 3.2. The head capsule
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Fig. 2. SEM micrographs of the larval heads of D. magna. (A) Dorsal view, fourth instar. (B) Lateral view, first instar. (C) Ventral view, fourth instar. (D) Magnification of the mouthparts of fourth instar. Abbreviations: ac, anteclypeus; amc, anterior mandibular acetabulum; ant, antenna; at, anterior tentorial pits; cas, cardo-stipes; ce, compound eye; cly, clypeus; cs, coronal suture; eb, egg burster; epi, epipharynx; fcs, frontoclypeal sulcus; fs, frontal sutures; gl, galea; lbr, labrum; lp, labial palp; md, mandible; mp, maxillary palp; pc, postclypeus; pm, postmentum; ppf; palpifer; prm, prementum; pt, posterior tentorial pit. Scale bars: (A) and (C) = 500 m; (B) and (D) = 200 m.
Fig. 3. Antenna of D. magna larvae, SEM micrographs. (A) First instar, latero-ventral view. (B) Second instar, ventral view. (C) Fourth instar, dorsal view. (D) Magnification of the pedicel of fourth instar larva in ventral view, showing the sensory pits on the apical half. Abbreviations: as, antennal sclerite; bs, basiconic sensilla; fl, flagellum; pe, pedicel; sc, scape; sp, sensory pits. Scale bars: (A) and (D) = 20 m; (B) = 50 m; (C) = 100 m.
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Fig. 4. Eipharynx of D. magna larvae, SEM micrographs. (A) First instar larva. (B) Third instar larva. (C) Fourth instar larva. (D) Magnification of the apical part of the epipharynx of first instar larva. Abbreviations: bs, basiconic sensilla; lr, labral setae; ps, palmate sensilla. Scale bars: (A) = 50 m; (B) and (C) = 100 m; (D) = 20 m.
and ventral sides of the apical third (Fig. 3A, B and D). The number of depressions varies in different instars, approximately 20 in the first instar and 30 in the fourth instar (Fig. 3A and D). The flagellum is considerably thin, shorter than the pedicel, with several sensilla basiconica at apex. The dorsal surface of the antenna is glabrous, lacking sensilla (Fig. 3C). 3.4. The mouthparts The mouthparts are of the typical mandibulate type. The labrum is trapezoid and slightly notched midapically, and articulated proximally with the anterior region of the clypeus (Fig. 2A and D). The labrum is furnished with two pairs of long labral setae along the distal margin, the inner pair nearly two-thirds the length of the outer pair (Fig. 2A). The membranous epipharynx is situated on the inner surface of the labrum and is basically in accordance with the labrum in shape (Fig. 4). At the mid-apical region appears a distinct longitudinal furrow (Fig. 4A–C). The epipharynx is furnished with dense lateral microtrichia pointed inward and sparse short medial spines pointed upward, with the apicolateral corner and longitudinal furrow regions glabrous (Fig. 4A–C). On the epipharynx, two types of sensilla are arranged symmetrically: five pairs of basiconic sensilla at the apical margin (three pairs) and between the microtrichial and spinous regions (two pairs), and two pairs of palmate sensilla near the basolateral microtrichial region (Fig. 4). A triangular sclerite connecting the epipharynx with the clypeus is enclosed with rows of spines. Several basiconic sensilla are observed at the basal and apical parts (Fig. 4A–C). The middle region enclosed by the spines is rugose and nearly glabrous. A few short microtrichia are extended along the midline of the triangular sclerite. The middle microtrichia nearly extend to two-thirds of the midline in the first instar, but do not reach the first third in the third and fourth instars (Fig. 4A–C). The paired mandibles are strongly sclerotized and curve inward, crossing each other apically and uniting to the subgena at the clypeal base (Fig. 2D). They are typically dicondylic, with a notched anterior ginglymus and knob-like posterior condyle (Fig. 5A–D).
The mandible tapers toward the apex, with two long subequal setae and one short seta on the lateral surface (Fig. 5B). The incisor region bears an acuminate triangular tooth, as well as a median and two basal teeth (Fig. 5A–D). The median tooth is longer than the basal teeth, but much shorter than the apical tooth. The molar region is equipped with numerous regularly arranged conical teeth, which are bordered by a row of spines on each lateral side (Fig. 5A–E). A tuft of branched microtrichia subtends the base of the molar region (Fig. 5E). In the newly hatched larva, the molar teeth and spines are sharp and intact, but become worn down after feeding (Fig. 5F). The paired maxillae each consist of the basal cardo-stipes, galea, lacinia, and three-segmented maxillary palp (Figs. 1D, 2C and 6A). The cardo-stipes is kidney-shaped and distally connected to the galea and lacinia medially and bears the palp laterally. Two setae are located on the cardo-stipes and palpifer, respectively (Figs. 1D and 2C). The galea is sclerotized in the basal half and membranous in the apical half, which is covered with dense microtrichia in the posterior side. The anterior surface of galea is equipped with numerous branched microtrichia, among which are distributed several basiconic sensilla at the apical half. The galea is broadly connected with the lacinia at base, the glabrous joint region bearing one palmate sensillum and two campaniform sensilla (Fig. 6A and B). The lacinia is considerably small, furnished with regularly arranged microplates at the apical half and dense microtrichia at the basal half. The three-segmented maxillary palp is inserted on a sclerotized palpifer, with the basal two segments roughly equal in length and shorter than the distal segment (Fig. 6A and B). The distal segment bears 14 basiconic sensilla on the apex (Fig. 6C). The labium is greatly reduced in size, with ligula absent. Most parts of the labium are retracted into the capsule; only a pair of two-segmented labial palps are prominently visible from the basal, mesially separated prementum between the cardo-stipes bases of the paired maxillae. Located at the base between the divided prementum is the opening of the salivary duct (Fig. 6D). The distal segment of the labial palp is much longer than the basal segment, approximately three times the length of the latter, and bears ten basiconic sensilla on its apex (Fig. 6D).
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Fig. 5. Mandibles of D. magna larvae, SEM micrographs. (A) Posterior view of the right mandible of first instar. (B) Anterior view of the left mandible of first instar. (C) Fronto-lateral view of the right mandible of first instar. (D) Fronto-lateral view of the left mandible of third instar. (E) Regular spines along the molar region and branched microtrichia at the base, first instar. (F) Molar teeth and spines worn down in fourth instar. Abbreviations: ant, antenna; c, condyle; g, ginglymus; ir, incisor region; mr, molar region. Scale bars: (A)–(C) and (F) = 50 m; (D) = 100 m; (E) = 20 m.
3.5. The thorax The prothorax is different from the meso- and metathorax by its prothoracic shield, paired spiracles, and chaetotaxy (Fig. 7A). The prothoracic shield is broad and saddle-like, with several short setae along the margin. The spiracle is situated at the posterolateral corner of the prothoracic shield and consists of a central atrial orifice and 12 peripheral apertures (Fig. 7A and C). Meso- and metathorax are similar in morphology and chaetotaxy, and lacking spiracles. The thoracic legs are 4-segmented: the coxa, femur, tibia and tarsus (Fig. 7D). The basal coxa and femur furnished with numerous microtrichia and long setae represent the thickest and longest segments, respectively. The apical halves of both the tibia and tarsus are hirsute (Fig. 7D). 3.6. The abdomen The abdomen consists of 11 segments and is furnished with short setae on the pinacula and erect subdorsal annulated processes inserted on stout basal protuberances (Fig. 1A). The long annulated processes are paired on segments I–IX (A1–A9), but single on the middorsal line of A10 (Figs. 1A and 7B). On the annulated process, numerous scaly cuticular processes are palmated. A campaniform sensillum is located at the base of the annulated process
(Fig. 7B). The first seven abdominal segments are roughly similar to each other in morphology and chaetotaxy (Fig. 1A). A8–A10 have much longer processes, and are thinner and differ considerably from others in chaetotaxy (Fig. 7B). Paired lateral spiracles and ventral prolegs are located on the first eight abdominal segments (Figs. 1A and 7B). The spiracle consists of seven spherical apertures (Fig. 7E), less than that of the prothoracic spiracle. The stout prolegs are unsegmented and are not arranged in a longitudinal line with the thoracic legs (Fig. 7F). A11 is reduced and remains as a four-lobed protrusile sucker (Fig. 7B). 3.7. The chaetotaxy of trunk The chaetotaxy of the mesothorax is similar to that of the metathorax, but different from that of the prothorax. For the abdomen, the first seven segments bear similar chaetotaxy, while segments VIII–X are individually distinct. The chaetotaxy of the trunk is illustrated in Fig. 8. Prothorax (T1): Along the anterior edge of the prothoracic shield are three clavate setae (XD1, XD2 and SD2, about 86, 104 and 99 m in length, respectively) and three punctures. Near the middorsal line appear a short setiform seta (D2) and a puncture. Two setae (D1 and SD1) are along the posterior edge of the shield. D1 is above the level of D2 and longer than half length of SD1. The distance
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Fig. 6. Maxilla and labium of D. magna larvae, SEM micrographs. (A) Maxilla of first instar, posterior view. Inset shows the magnification of the palmate sensillum. (B) Maxilla of third instar, showing the medial surface of the maxillary palp. Inset shows the magnification of one campaniform sensillum. (C) Magnification of the distal maxillary palp in first instar. (D) Labium region of first instar, showing the two-segmented labial palp. Abbreviations: bs, basiconic sensilla; cas, cardo-stipes; cs, campaniform sensilla; gl, galea; lc, lacinia; lp, labial palp; mp, maxillary palp; pm, postmentum; ppf; palpifer; prm, prementum; ps, palmate sensilla; sdo, salivary duct opening. Scale bars: (A) and (B) = 50 m; (C) = 10 m; (D) = 20 m.
between XD2 and SD2 is slightly shorter than that between D1 and SD1. L1 is on the lateral pinaculum, anteroventral to the spiracle. Two long setae (SV1 and SV2, 107 and 58 m long, respectively) and two microsetae (MSV1 and MSV2) on a crescent pinaculum dorsal to the coxal cavity. A spiculate ventral seta (V1) is close to the midventral line. Meso- and metathorax (T2 and T3): On the trapezoid dorsal pinaculum are three clavate setae (D1, D2, and SD1) and two microsetae (MD1 and MSD1). Ventral to the dorsal pinaculum are three pinacula, each of which is accompanied respectively by a long seta (SD2, L1, and L2) and a microseta (MSD2, ML1, and ML2). Subventral setae (SV1 and SV2) are located on two detached subventral pinacula. Ventral to the coxal cavity are one spiculate seta (V1) and a pair of microsetae (MV1 and MV2). Among these setae, SD1 is roughly 123 m long; longer than twice the length of D1. SD2 is slightly longer than L1, which is three-quarters as long as L2 (122 m). SV2 is nearly three-fourths the length of SV1. Abdominal segments I–VII (A1–A7): The annulated processes on A1–A7 measure 306 m in length on average. On the dorsal pinaculum are three rhabdiform setae (D1, D2, and SD1) and two microsetae (MD1 and MSD1). SD2 is accompanied by two microsetae (MSD2 and MSD3). Posterior to the spiracle is a long lateral seta (L1) with two microsetae (ML1 and ML3). L2 is located together with ML2 on a lower lateral pinaculum. Three setae (SV1–SV3) are on two subventral pinacula. Paired ventral setae (V1 and V2) are setiform, anterior to the proleg. D2 is almost equal to D1 in length. SD1 and SD2 are roughly in the same length (112 m on average), slightly shorter than L1. SV2 and SV3 are half as long as SV1. Abdominal segment VIII (A8): Three setae on the stout basal protuberance of the annulated process are rhabdiform, 41–42 m long for the lateral and mesal setae and 63 m for the posterior seta. L1 and L2 are rhabdiform. SV1–SV3 taper apically. L1 is about 128 m long, 1.6 times as long as L2 and markedly shorter than SV1. The setae between the spiracle and midventral line are identical to the preceding abdominal segments.
Abdominal segment IX (A9): Three setae on the basal protuberance of the annulated process are arranged similar to those on A8. D2, SD1, and SD2 are almost in the same length. L1 and SV1 are setiform, almost equal in length (173 m). V1 is about 63 m long. Abdominal segment X (A10): The protuberance of the single middorsal annulated process bears only two setae, each on lateral and mesal sides. A palmate pinaculum is located from the dorsal side to the subventral side, with one long, one short, and one minute seta at its posterior corner, regressively and four digitate lateral processes each terminated by a microseta. Setae D2, SD1 and SD2 are acicular, similar in length. 4. Discussion Larval data play an important role in the reconstruction of phylogenies in holometabolous insects, including Mecoptera (Beutel et al., 2009; Fraulob et al., 2012; Friedrich et al., 2013). In Panorpidae, although some species of Panorpa, Neopanorpa and Sinopanorpa have been described for their larval stages (Brauer, 1863; Byers, 1954, 1963, 1987; Cai and Hua, 2009b; Chen and Hua, 2011; Gassner, 1963; Mampe and Neunzig, 1965; Miyaké, 1912; Potter, 1938; Steiner, 1930; Yie, 1951), this paper is the first attempt to describe and illustrate the larva of the genus Dicerapanorpa. Panorpid larvae are peculiar among the holometabolous larvae for the presence of paired compound eyes (Byers, 1987). According to previous investigations, a compound eye comprises 24–35 ommatidia in Neopanorpa and Panorpa (Boese, 1973; Byers, 1963, 1987; Cai and Hua, 2009a,b; Steiner, 1930; Yie, 1951) and does not increase in the number of ommatidia during development, although differing among genera or species (Chen and Hua, 2011; Paulus, 1989; Yie, 1951). Based on our observations, the compound eye of D. magna larva is composed of over 40 ommatidia, similar to that of Sinopanorpa (Chen and Hua, 2011). The mandibulate mouthparts of panorpid larvae show slight differences among genera. For example, the labral apex is set with
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Fig. 7. Trunk of the first instar larvae of D. magna, SEM micrographs. (A) Lateral view of the thorax. (B) Lateral view of the abdominal segments VI to telson. Inset shows the magnification of the annulated process and setae on the abdominal segment IX. (C) Prothoracic spiracle. (D) Thoracic legs, lateral view. (E) Abdominal spiracle. (F) Prolegs of abdominal segments III and IV (A3 and A4), lateral view. Abbreviations: A, abdominal segment; ao, atrial orifice; ap, aperture; apr, annulated process; cs, campaniform sensillum; cx, coxa; fm, femur; pl, proleg; sp, spiracle; T, thorax; tb, tibia; tl, telson; ts, tarsus. Scale bars: (A), (B), (D) and (F) = 100 m; (C) and (E) = 10 m.
paired long setae laterally and one pair of short setae medially in Neopanorpa. In addition to the lateral paired long setae, two pairs of short setae are observed in D. magna and Sinopanorpa tincta and two or three pairs in Panorpa (Chen and Hua, 2011; Yie, 1951). According to Yie (1951), both the maxillary and labial palps bear two sensory cavities and several minute setae on the apices of the distal segments. Under SEM, we found the minute setae are in fact basiconic sensilla but the two sensory cavities were not observed. As gustatory chemoreceptors, the basiconic sensilla have a great ability to determine acceptable food items (Zacharuk and Shields, 1991). The number of basiconic sensilla on the apices of both maxillary and labial palps varies among different genera and species of Panorpidae. On the apex of the distal segment of the maxillary palp, 14 basiconic sensilla are present in D. magna, three more than that of Sinopanorpa, four more than that of Neopanorpa, and 6–8 more than that of Panorpa (Cai and Hua, 2009a,b; Chen and Hua, 2011; Yie, 1951). With respect to the segment number of labial palp in Panorpidae, controversial opinions are present. Bierbrodt (1942) regarded the labial palp as three-segmented based on the palpal muscles in Panorpa, while Hinton (1958) considered that the muscles referred by Bierbrodt (1942) were actually inserted on the lobed prementum and the labial palp was two-segmented. According to Snodgrass (1935), the prementum represents the united stipes of a pair of
maxilla-like appendages and is suggested often by a distal cleft between its stipital components. Our SEM observation supports Hinton (1958) with respect to the distally cleft prementum and the two-segmented labial palps. The distal segment of the labial palp bears apically ten basiconic sensilla in D. magna and S. tincta, eight or ten in Neopanorpa, and only six in Panorpa. The divided clypeal region of mecopteran larvae has been described in genera Panorpa and Sinopanorpa of Panorpidae (Bierbrodt, 1942; Chen and Hua, 2011), Apterobittacus, Harpobittacus, and Bittacus of Bittacidae (Applegarth, 1939; Currie, 1932; Tan and Hua, 2008), and in Boreus of Boreidae and Nannochorista of Nanonchoristidae (Beutel et al., 2009). Compared with the narrow and elongated anteclypeus in Bittacidae and other genera of Panorpidae, the anteclypeus in Dicerapanorpa is broader, with a distinct transverse demarcation, similar to that of Nannochorista (Beutel et al., 2009). Dicerapanorpa larvae can be readily distinguished from other genera of Panorpidae by the following characters: (i) the compound eye is composed of more than 40 ommatidia; (ii) the numbers of the basiconic sensilla on the apices of both maxillary and labial palps are 14 and 10, respectively; (iii) the setae are short and annulated processes are longer; (iv) MSD1 on the prothorax is located almost along the same horizontal line with SD2; and (v) microtrichia are lacking posterior to SD2 on A10.
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Fig. 8. Chaetotaxy of the trunk of D. magna, first instar. Abbreviations: A, abdomen; D, dorsal setae; L, lateral setae; MD, minute dorsal seta; ML, minute lateral setae; MSD, minute subdorsal setae; MSV, minute subventral seta; MV, minute ventral setae; pl, proleg; SD, subdorsal setae; sp, spiracle; SV, subventral setae; T, thorax; V, ventral setae; XD, prothoracic setae. Scale bar = 0.1 mm.
The abdomen of D. magna larvae terminates in a four-lobed protrusile sucker, which is similar to that of the rectal processes of other eruciform larvae of Pistillifera, but may not be homologous with the anal papillae in Nannochorista because of the very farreaching structural and functional differences (Beutel et al., 2009; Pilgrim, 1972). According to embryonic evidence, the protrusile sucker of Panorpa is developed from the processes of the eleventh abdominal segment (Du et al., 2009). Hence, the reduced A11 in Panorpidae may be evolved secondarily, similar to the remarkable A11 in Boreidae (Byers, 1987; Russell, 1982). Mecoptera exhibit remarkable morphological diversity in their larval stage, and adapted to various habitats. The larvae of the primitive Nannochoristidae are campodeiform, with their body surface almost smooth and shining, adapted to aquatic habits (Fraulob et al., 2012; Pilgrim, 1972). The larvae of Boreidae and Panorpodidae were regarded as scarabaeiform, and only bear short setae on their trunks, relating to tunneling into the sod of host mosses and substrate, respectively (Byers, 1987, 1997; Cooper, 1974; Penny, 1977; Peterson, 1951; Potter, 1938; Russell, 1982; Suzuki, 1990). The larvae of Bittacidae and Panorpidae are eruciform and bear prominent protuberances on their trunk (Byers, 1987). The protuberances
in Bittacidae have two or three branches from which setae arise (Applegarth, 1939; Currie, 1932; Setty, 1939, 1940; Suzuki, 1990; Tan and Hua, 2008, 2009), but the protuberances in Panorpidae each bear a long annulated process (Brauer, 1863; Byers, 1954, 1963; Cai and Hua, 2009b; Chen and Hua, 2011; Gassner, 1963; Mampe and Neunzig, 1965; Miyaké, 1912; Potter, 1938; Steiner, 1930; Yie, 1951). The newly hatched larvae of Bittacidae do not burrow into soil and usually cover their body surfaces with mud, probably for camouflage (Tan and Hua, 2008). However, the larvae of Panorpidae burrow through the soil shallowly (approximately 2.5–7.5 cm) to forage beneath potential food. When the food is situated over 7.5 cm away, they crawl above the substrate until food is located, and then constructs a new burrow for concealment while feeding (Mampe and Neunzig, 1965). We speculate that the occurrence of the long annulated processes on the larval trunk of Panorpidae is associated with the above-substrate searching and fossorial feeding habits, and is very likely evolved as adaptations to protect the soft body from much leaf litter. The dense palmate cuticular processes of the annulated process may reduce friction when the larvae move forward either above or below the substrate.
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